The present invention relates to synthetic pyrazinium compounds and their use in photoreactive enzymatic systems. More specifically, the pyrazinium compounds of the invention are useful as electron carriers in the photoenzymatic production of hydrogen and ammonia.
Photosynthesis is a process that is fundamental for the subsistence of our Biosphere. When carried out by living organisms, this transduction of light energy into chemical energy requires the pigment chlorophyll as a photosensitizer. In higher order organisms, such as eucaryotic organisms, this function is performed in specialized cell organelles, the chloroplasts, which possess many properties like those of an independent organism.
Light energy absorbed by the chloroplasts results in photolysis of water and a potential increase of the photolytically generated "low-energy" electrons (+800 mV) to a "high-energy" value of -600 mV. The "high energy" electrons are trapped by the primary electron carriers found in the chloroplasts. The resulting energy-rich, reduced carriers are then used by the organism to convert carbon dioxide into carbohydrates or hydrocarbons, and nitrogen into ammonia using enzymatically-linked processes. Moreover, under conditions wherein these processes are not fully operative, such as limited access to carbon dioxide or nitrogen, the reduced electron carriers are diverted to a hydrogen production reaction catalyzed with nitrogenase and hydrogenases.
Although the photosynthetic production and utilization processes occurring in chlorophyll organelles are not completely understood, it is known that successive absorption of two light quanta by the coupled chlorophyll pigments P.680 and P.700 is needed in order to raise the energy of electrons produced from water photolysis to that required to produce reduced electron carriers. The high potentials reached by the photoexcited electrons are sufficient to reduce the iron-sulfur clusters present in bound primary electron carriers such as ferredoxin. The energy is then transferred to soluble electron carriers such as free ferredoxin or flavodoxin. Further discussion of the biological photosynthetic process may be found in J. R. Benemann, et. al., "Advances in Microbial Physiology", Vol. 5, Academic Press London, 1971, pp. 135-172; D. I. Arnon, et al., Proc. Nat. Acad. Sci. USA, 78, 2942-6 (1981); M. Calvin, in "Living Systems As Energy Converters", North Holland Pub., Amsterdam, 1977, pp. 231-259.
Proteins such as ferredoxins and flavodoxins are the natural electron carriers present in biological organisms that participate in the in vivo transfers of high energy electrons in both aerobic and anaerobic processes, see E. J. Knight, et al., J. Biol. Chem., 241, 2752 (1966). These proteins participate in in vivo light dependent nitrogen fixation, carbohydrate production and hydrogen evolution as well as light independent anaerobic nitrogen fixation, see T. R. Hamilton, et al., Proc. Natl. Acad. Sci. USA, 52, 637 (1964). Essentially, they carry the high energy electrons from chlorophyll to the enzymes which use them.
Artificial systems using ferredoxins and flavodoxins have been developed recently as part of several investigations of the synthetic production of hydrogen by photolysis of water. Such systems have also been utilized as test assay models for the study of photosynthetic reactions. Typically, the system can employ a synthetic photo-activator or isolated plant chloroplasts, an electron carrier and an enzyme such as nitrogenase or hydrogenase, see J. R. Benemann, et al., Proc. Nat. Acad. Sci. USA, 64, 1079 (1969); and J. R. Benemann in "Living Systems As Energy Converters", North-Holland Publ., Amsterdam, 1977, pp. 285-297. In such a model, for example, the activity of the system stimulated by the photosynthetic reaction is followed by the reduction of acetylene to ethylene.
Studies using isolated chloroplast systems have shown that other compounds can function as electron carriers and can be substituted for ferredoxin or flavodoxin. For example, dipyridyls such as methyl viologen, benzyl viologen and cyclic analogs thereof are able to couple illuminated chloroplasts and the enzyme hydrogenase, see K. K. Rao, et al., in "Photosynthesis In Relation to Model Systems," pp. 299-329, Elsevier, Amsterdam, 1979; I. Okura, et al., J.C.S. Chem. Comm., 1980, 84. These synthetic compounds can also interact in cellular photochemical redox reactions to cause "short circuiting" of the photosynthetic pathways. It is not surprising, therefore, to find that a few dipyridyls such as diquat and paraquat have herbicidal activity, see B. Kock, et al., Biochem Biophys. Acta, 1091, 347 (1965).
Generally, however, very few organic compounds are known to function effectively as electron carriers for chloroplast or synthetic photo-activator systems. Typically, the potentials of the reduced forms of known low molecular weight organic carriers do not match the potential required for effective enzyme coupling. As a consequence, transfer of the energetic electrons by these carriers becomes inefficient. Moreover, any tendency of the reduced forms of the carriers to remain bound to the chloroplast favors the undesirable reverse reaction with the organelle which will cause failure of the electron transfer process. Synthetic electron carriers designed and synthesized from protein material, such as synthetic analogs of flavodoxin or ferredoxin, are also theoretically possible. Their syntheses, however, would be complicated, their half lives short and they would require special process and synthetic measures incident to the use of proteins. Accordingly, the nature of the carrier is one of the limiting features of an artificial photosynthetic system.
It is, therefore, an object of the invention to develop synthetic organic compounds which can function as efficient electron carriers in a chloroplast or synthetic photo-activator photosynthetic system. Another object is the production of a stable organic compound which will increase the efficiency of a photosynthetic system. A further object is the production of an organic compound which is highly stable in a reduced, energetic state. Yet another object is the production of water-soluble organic compounds which maximize the transduction process utilizing the chloroplast organelle.